Protein glycosylation has been considered as one of the most significant protein modifications. It has been widely recognized that glycosylation is associated with disease progression, such as cancer, heart failure, and other congenital disorders. The investigation of glycoproteins and their associated glycans is the key to understanding glycoprotein functions in biological pathways and disease development as well as biomarker discovery. To this end, the present inventors previously developed the solid-phase extraction of glycopeptides (SPEG) for capture of glycosylated peptides, which has been widely applied to both quantitative analysis of glycoproteins and identification of glycosylation sites. In this method, glycosylated peptides from digested glycoproteins are captured by using hydrazide beads after glycans on glycopeptides are oxidized. Following the removal of non-glycosylated peptides, these glycosylated peptides are then enzymatically released from the solid support for mass spectrometry (MS) analysis. Using this method, thousands of new N-linked glycosylation sites have been identified. However, the glycans are removed from glycopeptides during the capture processes and their structures are not identified.
Quantitative and qualitative characterization of glycoprotein associated glycans is thus biologically significant. Many efforts have been devoted to derivatizing glycans at their reducing ends, on sialic acids, or permethylation of hydroxyl groups. Quantification by modifying glycans at their reducing ends with fluorescent tags has advantages including high sensitivity and high throughput, but the detection resolution is limited by front-end separation techniques. Stable isotope-coded tags have been developed and shown excellent resolution when combined with LC-MS analysis. Yet most isotope tags are mass-shift based, which may complicate spectra and lead to difficulty in data analysis. Isobaric tags, on the other hand, do not introduce more peaks on MS spectra and make it possible to analyze multiple samples concurrently.
Therefore, there still exists an unmet need to develop novel isotopic tags and analytical methods which can specifically isolate glycans or glycopeptides from complex mixtures, including biological and clinical samples.